WO2017007006A1 - Récipient destiné à être utilisé pour la cryoconservation de sperme et procédé de production correspondant, procédé de cryoconservation de sperme, et procédé de fécondation in vitro - Google Patents

Récipient destiné à être utilisé pour la cryoconservation de sperme et procédé de production correspondant, procédé de cryoconservation de sperme, et procédé de fécondation in vitro Download PDF

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Publication number
WO2017007006A1
WO2017007006A1 PCT/JP2016/070194 JP2016070194W WO2017007006A1 WO 2017007006 A1 WO2017007006 A1 WO 2017007006A1 JP 2016070194 W JP2016070194 W JP 2016070194W WO 2017007006 A1 WO2017007006 A1 WO 2017007006A1
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sperm
container
gel
gel shell
cryopreservation
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PCT/JP2016/070194
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English (en)
Japanese (ja)
Inventor
慎司 境
田谷　正仁
和尚 冨田
美彦 細井
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国立大学法人大阪大学
学校法人近畿大学
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Publication of WO2017007006A1 publication Critical patent/WO2017007006A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00—Instruments or methods for reproduction or fertilisation
    • A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination

Definitions

  • the present invention relates to a sperm cryopreservation container and a method for producing the same, a sperm cryopreservation method, and an in vitro fertilization method.
  • Patent Documents 1 to 4 and Non-Patent Documents 1 to 3 describe containers for cryopreserving sperm.
  • ICSI microinsemination
  • Intracytoplasmic Sperm Injection it was difficult to freeze and store the very few spermatozoa with high efficiency.
  • the present invention has been made in view of the above problems, and a sperm cryopreservation container capable of effectively performing cryopreservation and recovery of the sperm even when the number of sperm is small and its production
  • Another object is to provide a method, a sperm cryopreservation method, and an in vitro fertilization method.
  • a sperm cryopreservation method for solving the above problems includes the following (a) to (f): (a) a gel shell containing glycosaminoglycan chains and / or cellulose chains, Providing a container having a liquid core coated with a gel shell; (b) injecting sperm into the liquid core of the container to obtain the container holding the sperm; (c) (D) thawing the container after cryopreservation; (e) decomposing the glycosaminoglycan chain from the gel shell of the container after thawing; And / or an enzyme that decomposes the cellulose chain to decompose at least a part of the gel shell; and (f) the sperm held in the container after the gel shell is decomposed.
  • a sperm cryopreservation method capable of effectively performing cryopreservation and recovery of sperm even when the number of sperm is small.
  • the gel shell may contain hyaluronic acid chains, and the gel shell of the container after thawing may be treated with an enzyme that degrades the hyaluronic acid chains.
  • the in vitro fertilization method for solving the above problems includes the following (a) to (g): (a) a gel shell containing glycosaminoglycan chains and / or cellulose chains, and the gel Providing a container having a liquid core covered with a shell; (b) injecting sperm into the liquid core of the container to obtain the container holding the sperm; (c) the sperm (D) thawing the container after cryopreservation; (e) decomposing the glycosaminoglycan chain in the gel shell of the container after thawing; Treating with an enzyme and / or an enzyme that degrades the cellulose chain to degrade at least a portion of the gel shell; (f) recovering the sperm retained in the container after the gel shell has been degraded.
  • the in vitro fertilization may be microinsemination.
  • a container for sperm cryopreservation according to an embodiment of the present invention for solving the above-described problem holds a gel shell containing glycosaminoglycan chains and / or cellulose chains, and sperm coated on the gel shell. And a liquid wick. According to the present invention, it is possible to provide a sperm cryopreservation container capable of effectively performing cryopreservation and recovery of sperm even when the number of sperm is small.
  • the gel shell may contain hyaluronic acid chains.
  • the average diameter of the container may be 10 ⁇ m or more and 3000 ⁇ m or less.
  • the container for sperm cryopreservation may be used in any one of the sperm cryopreservation methods or any one of the in vitro fertilization methods.
  • a method for producing a sperm cryopreservation container according to an embodiment of the present invention for solving the above-described problem is a method for producing a sperm cryopreservation container having a gel shell and a liquid core coated on the gel shell.
  • the gel shell may contain a hyaluronic acid chain.
  • a sperm cryopreservation container and a method for producing the same, a sperm cryopreservation method, and an in vitro fertilization method that can effectively perform cryopreservation and recovery of the sperm even when the number of sperm is small Can be provided.
  • Example 1 which concerns on one Embodiment of this invention, it is explanatory drawing which shows the result of having evaluated the influence which a degradation enzyme has on the survival rate of a sperm.
  • Example 1 which concerns on one Embodiment of this invention, it is explanatory drawing which shows the result of having evaluated the influence which a degradation enzyme exerts on the motility rate of a sperm.
  • Example 2 which concerns on one Embodiment of this invention, it is explanatory drawing which shows the result of having evaluated the collection
  • Example 3 which concerns on one Embodiment of this invention, it is explanatory drawing which shows the result of having examined the recovery method of the sperm from the container for sperm cryopreservation.
  • the sperm cryopreservation method comprises the following (a) to (f): (a) a gel shell containing glycosaminoglycan chains and / or cellulose chains, and a liquid core coated on the gel shell. (B) Injecting sperm into the liquid core of the container to obtain the container holding the sperm; (c) Freezing and storing the container holding the sperm (D) Thawing the container after cryopreservation; (e) Degrading the gel shell of the container after thawing, degrading the glycosaminoglycan chain and / or the cellulose chain Treating with an enzyme that degrades at least a portion of the gel shell; and (f) recovering the sperm retained in the container after degradation of the gel shell.
  • the in vitro fertilization method has the following (a) to (g): cocoon (a) a gel shell containing glycosaminoglycan chains and / or cellulose chains, and a liquid core coated on the gel shell. Preparing a container; (b) injecting sperm into the liquid core of the container to obtain the container holding the sperm; (c) cryopreserving the container holding the sperm; (D) thawing the container after cryopreservation; (e) decomposing the glycosaminoglycan chain and / or the cellulose chain from the gel shell of the container after thawing. Treating with an enzyme to degrade at least a portion of the gel shell; (f) recovering the sperm retained in the container after decomposition of the gel shell; and (g) recovered. Perform in vitro fertilization using the sperm And; including.
  • a sperm cryopreservation container (hereinafter referred to as “this container”) holds a gel shell containing glycosaminoglycan chains and / or cellulose chains, and sperm coated on the gel shell.
  • This container holds a gel shell containing glycosaminoglycan chains and / or cellulose chains, and sperm coated on the gel shell.
  • a liquid core A sperm cryopreservation container
  • the prepared container is the one that has not yet retained sperm.
  • This container may be prepared in advance by preparing a pre-manufactured container (for example, purchasing and preparing the container if the container is in the market) May be manufactured.
  • FIG. 1A shows a cross section of an example of the container.
  • the container 10 has a spherical shape and includes a gel shell 11 and a liquid core 12 covered with the gel shell 11.
  • the gel shell of this container contains glycosaminoglycan chains and / or cellulose chains. That is, the gel shell may include a glycosaminoglycan chain, may include a cellulose chain, or may include a glycosaminoglycan chain and a cellulose chain. When the gel shell includes glycosaminoglycan chains, the gel shell may further include cellulose chains. When the gel shell includes cellulose chains, the gel shell may further include glycosaminoglycan chains.
  • glycosaminoglycan chain is a polymer chain of glycosaminoglycan.
  • Glycosaminoglycans are polysaccharides composed of disaccharides composed of amino sugar (galactosamine or glucosamine) and uronic acid (glucuronic acid or iduronic acid) or galactose repeatedly bonded.
  • the glycosaminoglycan is selected from, for example, hyaluronic acid, chondroitin sulfate (one or more selected from the group consisting of chondroitin sulfate A, chondroitin sulfate B and chondroitin sulfate C), keratan sulfate, heparan sulfate and heparin.
  • hyaluronic acid is particularly preferable. That is, the gel shell particularly preferably contains a hyaluronic acid chain.
  • the cellulose chain is a polymer chain of cellulose.
  • Cellulose is a polysaccharide composed of ⁇ -glucose repeatedly bonded linearly by glycosidic bonds.
  • the cellulose chain may be a cellulose chain contained in a cellulose derivative. That is, in this case, the gel shell includes a cellulose derivative, and the cellulose derivative includes a cellulose chain.
  • a cellulose derivative is a polymer other than cellulose, which is formed by introducing a functional group into a cellulose chain and includes the cellulose chain and the functional group.
  • the cellulose derivative may be one or more selected from the group consisting of, for example, nitrocellulose, acetylcellulose, and cellulose ether (for example, carboxymethylcellulose).
  • the gel shell is composed of a polymer gel containing glycosaminoglycan chains and / or cellulose chains as described above.
  • the content of glycosaminoglycan chains and / or cellulose chains in the polymer constituting the gel shell is such that the gel shell can be degraded by a degrading enzyme to the extent that sperm can be recovered in (e) above.
  • it is preferably 30% by weight or more, more preferably 50% by weight or more, and particularly preferably 70% by weight or more.
  • the polymer containing a glycosaminoglycan chain and not containing a cellulose chain is the glycosaminoglycan.
  • the polymer that contains the cellulose chain and does not contain the glycosaminoglycan chain means that the polymer contains 30% by weight or more of the cellulose chain.
  • the polymer containing both the chains means that the glycosaminoglycan chain and the cellulose chain are contained in a total of 30% by weight or more.
  • the gel shell may further contain other components in addition to the glycosaminoglycan chain and / or the cellulose chain.
  • the other components are not particularly limited as long as they do not prevent the formation of the gel shell in the production of the container described later.
  • one or more selected from the group consisting of gelatin, collagen, and polyphenols may be used. Good.
  • the liquid core of this container is composed of liquid.
  • the liquid which comprises a liquid core will not be restricted especially if a sperm can survive in it. That is, the liquid constituting the liquid core is, for example, a liquid having an osmotic pressure and pH suitable for sperm survival, and may further include a nutrient source necessary for sperm movement.
  • This container is used for the sperm cryopreservation method and in vitro fertilization method according to this embodiment. That is, this embodiment includes the use of the container for a sperm cryopreservation method and the use of the container for an in vitro fertilization method. In addition, the present embodiment includes a sperm cryopreservation method using the container and an in vitro fertilization method using the container.
  • the size of the container is not particularly limited as long as it is in a range suitable for use in the sperm cryopreservation method and in vitro fertilization method according to the present embodiment, but is preferably a size similar to an egg cell or a zona pellucida thereof.
  • the average diameter of the container may be 10 ⁇ m or more and 3000 ⁇ m or less, preferably 30 ⁇ m or more and 2000 ⁇ m or less, more preferably 50 ⁇ m or more and 1000 ⁇ m or less, and 50 ⁇ m or more and 500 ⁇ m or less. It is particularly preferred.
  • the average diameter of the container is the average diameter of the gel shell.
  • the average thickness of the gel shell of the container is not particularly limited as long as it is in a range suitable for use in the sperm cryopreservation method and in vitro fertilization method according to the present embodiment. For example, 0.01 ⁇ m or more and 1400 ⁇ m The thickness may be 1 ⁇ m or more and 1000 ⁇ m or less, more preferably 10 ⁇ m or more and 500 ⁇ m or less, and particularly preferably 10 ⁇ m or more and 100 ⁇ m or less.
  • the manufacturing method of the container for sperm cryopreservation (this container) which concerns on this embodiment is a manufacturing method of the container for sperm cryopreservation which has a gel shell and the liquid core coat
  • the gel core formed in (x) above is not particularly limited as long as it does not contain sperm and is composed of a gel that can be dissolved in (z) above. That is, the gel core first prepares a precursor solution that gels under predetermined conditions (for example, one or more selected from the group consisting of temperature, pH, light irradiation and contact with other components), and then It can form by applying the said precursor solution to the said predetermined conditions, and gelatinizing.
  • predetermined conditions for example, one or more selected from the group consisting of temperature, pH, light irradiation and contact with other components
  • the gel core is, for example, one or more selected from the group consisting of gelatin, alginic acid, cellulose, pectin, amylopectin, hyaluronic acid, collagen, keratin or konjac mannan gel, gels of these derivatives and matrigel. It may be configured.
  • Formation of the gel shell according to the above (y) is performed by a crosslinking reaction of a precursor polymer containing a glycosaminoglycan chain and / or a cellulose chain and a crosslinkable functional group. That is, for example, when the gel shell includes a hyaluronic acid chain, the gel shell is formed by a crosslinking reaction of a precursor polymer (hyaluronic acid derivative) including the hyaluronic acid chain and a crosslinkable functional group.
  • the precursor polymer can be obtained by introducing a crosslinkable functional group into a polymer containing a glycosaminoglycan chain and / or a cellulose chain.
  • the precursor polymer containing a glycosaminoglycan chain and a crosslinkable functional group includes, for example, a polymer containing the glycosaminoglycan chain and a compound containing a functional group corresponding to the crosslinkable functional group Is produced by chemically or enzymatically reacting.
  • the precursor polymer containing a cellulose chain and a crosslinkable functional group is obtained by, for example, chemically reacting a polymer containing the cellulose chain and a compound containing a functional group corresponding to the crosslinkable functional group. Generated.
  • the crosslinkable functional group contained in the precursor polymer is not particularly limited as long as it contributes to the formation of the gel shell.
  • a hydroxyl group, amino group, carboxyl group, aldehyde group, thiol group, phenol group, epoxy As at least one selected from the group consisting of a group, acryloyl group, vinyl group, benzophenone group, diazoester group, arylazide group, diazirine group, methacrylate group, allyl group, ethylenically unsaturated group, and hydrazide group Also good.
  • the gel shell formed by the crosslinking reaction of the precursor polymer is composed of a polymer containing a glycosaminoglycan chain and / or a cellulose chain and a crosslinking group.
  • the gel core covered with the gel shell is not decomposed and the gel core is dissolved (for example, temperature change, pH change, light It is carried out by treatment with irradiation and one or more selected from the group consisting of contact with other components (for example, enzymes and / or chelating agents).
  • the gel core when the gel core is composed of a gelatin gel, the gel core is treated with a proteolytic enzyme (for example, trypsin) or heated to a temperature at which the gelatin gel is redissolved.
  • the core can be dissolved.
  • the gel core when the gel core is composed of an alginate gel, the gel core can be dissolved by treating the gel core with a chelating agent (for example, citric acid) and / or an alginate lyase.
  • a chelating agent for example, citric acid
  • the liquid core covered with the gel shell is formed by dissolution of the gel core covered with the gel shell. That is, by sequentially performing the above (x), (y), and (z), this container having a gel shell and a liquid core coated on the gel shell is obtained.
  • the method of injecting sperm into the liquid core of the container is not particularly limited as long as it is a method capable of injecting sperm outside the container into the liquid core through the gel shell, It is preferred to use an injection pipette.
  • FIG. 1B shows an example in which the sperm S is injected into the liquid core 12 of the container 10 using the injection pipette 20. That is, injecting the sperm S into the liquid core 12 first holds the sperm S inside the injection pipette 20, and then uses the tip of the injection pipette 20 holding the sperm S as the gel shell of the container 10. 11 is inserted and then penetrated, and then the sperm S in the injection pipette 20 is discharged into the liquid core 12.
  • the injection pipette is not particularly limited as long as it is a hollow needle-like member capable of holding sperm inside, but those used for in vitro fertilization (for example, microinsemination) are preferably used.
  • An injection pipette having an inner diameter of 4 ⁇ m to 10 ⁇ m is preferably used.
  • the injection pipette is preferably made of glass or transparent resin.
  • the container 10 when using an injection pipette, it is preferable to hold the container 10 by a holding pipette 30 as shown in FIG. 1B. That is, in this case, first, the main container 10 is held at the tip of the holding pipette 30 and then the liquid core of the main container 10 held by the holding pipette 30 using the injection pipette 20 as described above. 12 is injected with sperm S.
  • the holding pipette is not particularly limited as long as it is a hollow rod-like member capable of holding the container at the tip thereof, but those used for in vitro fertilization (for example, microinsemination) are preferably used.
  • a holding pipette having an inner diameter of 15 to 30 ⁇ m is preferably used.
  • the holding pipette is preferably made of glass or transparent resin.
  • the injection of sperm into the liquid core of the container according to the above (b) is preferably performed by the same operation as the operation of injecting sperm into the egg cell in microinsemination.
  • an embryo culturer accustomed to the operation of microinsemination can efficiently and surely inject sperm into this container.
  • the sperm held in the container is naturally a live sperm.
  • the origin of the sperm retained in the container is not particularly limited, and may be human sperm, or sperm of a non-human animal (eg, mouse, rabbit, rat, monkey, cow, horse, sheep or pig). May be.
  • the present invention is particularly useful for cryopreservation of rare sperm and in vitro fertilization using rare sperm after cryopreservation.
  • the present invention relates to human sperm, particularly rare sperm from human patients with oligospermia or azoospermia (eg, sperm collected from semen of male patients with oligospermia, or azoospermia This is useful when using sperm collected from the testes of male patients.
  • the method for cryopreservation according to the above (c) is not particularly limited as long as the sperm retained in the container can be frozen and stored viable after thawing. That is, for example, first put this container holding sperm in a cell cryopreservation container (for example, a commercially available cryopreservation instrument (Cryotop (registered trademark), manufactured by KITAZATO CORPORATION)), then using liquid nitrogen, By maintaining the container at a low temperature suitable for cryopreservation (eg, ⁇ 196 ° C. or lower), the sperm and liquid core in the container are frozen. As a result, as shown in FIG. 1C, the present container 10 including the frozen sperm S and the liquid core 12 is obtained.
  • the time for cryopreservation is not particularly limited, and is appropriately determined according to the time of performing in vitro fertilization using sperm after thawing.
  • the method of thawing according to the above (d) is not particularly limited as long as the sperm cryopreserved in this container can be maintained in a alive state even after thawing. That is, for example, the sperm is thawed by immersing the container after cryopreservation in a solution at a temperature suitable for sperm survival (for example, 37 ° C.). As a result, as shown in FIG. 1D, the container 10 having the liquid core 12 containing the live sperm S and the gel shell 11 covering the liquid core 12 is obtained as before the cryopreservation.
  • the enzyme used for decomposing the gel shell according to (e) above is not particularly limited as long as it decomposes glycosaminoglycan chains and / or cellulose chains contained in the gel shell. That is, when the gel shell includes a hyaluronic acid chain, an enzyme (for example, hyaluronidase) that degrades the hyaluronic acid chain is used. When the gel shell includes a chondroitin sulfate chain, an enzyme (for example, chondroitinase) that degrades the chondroitin acid chain is used. When the gel shell contains keratan sulfate chains, an enzyme (for example, keratanase) that degrades the keratan acid chains is used.
  • the gel shell includes heparan sulfate chains and / or heparin chains
  • an enzyme for example, heparinase
  • an enzyme for example, cellulase
  • decomposes the cellulose chains is used.
  • the gel shell contains a hyaluronic acid chain
  • the gel shell of the container after thawing is converted into an enzyme that decomposes the hyaluronic acid chain (for example, , Hyaluronidase).
  • the decomposition of the gel shell it is possible to recover the sperm retained in the liquid core of the container (for example, the sperm retained in the container using an injection pipette without penetrating the gel shell). At least a part of the gel shell may be decomposed to such an extent that the sperm retained in the container can be recovered or the sperm retained in the container can come out of the container.
  • FIG. 1E shows that the gel shell 11 of the container disappears due to enzymatic decomposition (broken line), and the live sperm S held in the liquid core 12 of the container can be easily recovered.
  • the time required for the enzymatic degradation of the gel shell varies depending on the amount of enzyme used and conditions such as temperature, but is preferably as short as possible, for example, preferably 30 minutes or less, and preferably 10 minutes or less. It is particularly preferred that
  • the sperm collection method according to the above (f) is not particularly limited as long as it is a method capable of collecting live sperm released by enzymatic decomposition of the gel shell in a state suitable for subsequent use.
  • an injection pipette may be used in the collection of sperm.
  • FIG. 1F shows an example of how the sperm S is collected using the injection pipette 20.
  • the in vitro fertilization according to (g) is further performed using the sperm collected in (f) above. That is, in vitro, sperm collected from the container is brought into contact with egg cells collected in advance for use in in vitro fertilization to produce a fertilized egg.
  • FIG. 1G shows an example of microinsemination.
  • microinsemination is performed under the microscope with the tip of the injection pipette 20 holding the sperm S inside the cytoplasm C through the zona pellucida Z of the egg cell E held by the holding pipette 30. It is performed by inserting and releasing sperm S into the cytoplasm C from the injection pipette 20.
  • injection of sperm into the container is performed using an injection pipette
  • the gel shell of the container after thawing is decomposed by enzymatic treatment
  • the sperm state is maintained well by an engineer (for example, an embryo cultivator) accustomed to the operation of the injection pipette.
  • an engineer for example, an embryo cultivator
  • cryopreservation, recovery, and application to in vitro fertilization of the sperm can be performed efficiently and reliably.
  • the effects of an enzyme that degrades glycosaminoglycan chains and an enzyme that degrades cellulose chains on sperm viability and motility were evaluated.
  • a commercially available enzyme was used as the degrading enzyme. That is, hyaluronidase (Irvine) was used as the hyaluronic acid chain degrading enzyme. Cellulase (manufactured by Worthington Biochemical) was used as the cellulose chain degrading enzyme. Also, alginate lyase (manufactured by Sigma) which is an alginate degrading enzyme was used.
  • sperm collected from a human male patient but not used in human in vitro fertilization was used. That is, spermatozoa collected for in vitro fertilization but not used at the medical corporation Sankeikai IVF Namba Clinic (Osaka Prefecture, Japan) were used after obtaining informed consent and obtaining consent.
  • a solution containing 4 ⁇ 10 6 cells / mL sperm and a degrading enzyme 40 IU / mL hyaluronidase, 1 mg / mL cellulase, or 0.2 mg / mL alginate lyase
  • a degrading enzyme 40 IU / mL hyaluronidase, 1 mg / mL cellulase, or 0.2 mg / mL alginate lyase
  • sperm viability was assessed by a hypotonic liquid tail swelling test. That is, 50 ⁇ L of physiological saline containing 4 ⁇ 10 6 cells / mL sperm was dropped into 500 ⁇ L of hypotonic solution (150 mOsm), and incubated at room temperature for 30 minutes. Thereafter, sperm in which tail swelling was confirmed was evaluated as a live sperm. Then, under the microscope, the number of viable spermatozoa contained in 100 spermatozoa was counted, and the number of viable spermatozoa was defined as the sperm viability (%).
  • the sperm motility rate is determined by counting the number of sperm moving in the visual field under a microscope using a McLa chamber (SEFI-MEDICAL INSTRUMENT), The ratio of the number of sperm was calculated as sperm motility rate (%).
  • FIGS. 2A and 2B show the results of evaluating the survival rate and motility rate of sperm, respectively.
  • the viability and motility of spermatozoa in contact with any of hyaluronidase, cellulase and alginate lyase were not significantly different from those of spermatozoa not in contact with the enzyme, and the statistically significant difference was Not observed (Turkey-kramer method). That is, it was confirmed that hyaluronidase, cellulase, and alginate lyase have no effect on sperm that would reduce their survival rate and motility rate.
  • a hyaluronic acid derivative (crosslinkable hyaluronic acid) was produced by chemically introducing a phenol group as a crosslinkable functional group into hyaluronic acid (molecular weight: about 1 million, manufactured by JNC). That is, hyaluronic acid was dissolved in 30 mM 2-Morpholinoethanesulfide acid, monohydrate (MES) buffer (pH 6) to a concentration of 0.1% (w / v), and then tyramine hydrochloride (manufactured by Tokyo Chemical Industry Co., Ltd.) was further added. It was dissolved to 0.5% (w / v).
  • MES 2-Morpholinoethanesulfide acid, monohydrate
  • tyramine hydrochloride manufactured by Tokyo Chemical Industry Co., Ltd.
  • the obtained solution was further added with water-soluble carbodiimide (manufactured by Peptide Institute) and hydroxysuccinimide (manufactured by Wako Pure Chemical Industries, Ltd.) of 0.47% (w / v) and 0.27% (w / v), respectively. ) And stirred at room temperature for 20 hours. Then, after dialysis in purified water using a dialysis membrane (fractionated molecular weight of 10,000 to 20,000), the solution is freeze-dried to obtain a hyaluronic acid derivative containing a hyaluronic acid chain and a phenol group. Got.
  • a double tube structure was formed by arranging a 26G syringe in a 21G (gauge) syringe.
  • the 26G syringe was placed so that the tip of the 26G syringe was placed in the 21G syringe.
  • a 7.5% (w / v) gelatin solution is flowed into the 26G syringe at a flow rate of 4.5 mL / h, and liquid paraffin is flowed between the 26G syringe and the 21G syringe at a flow rate of 4.2 mL / min.
  • the gelatin solution was allowed to flow into the liquid paraffin from the tip of the 26G syringe.
  • the liquid paraffin containing droplets of the gelatin solution thus obtained was cooled in ice water to form spherical gelatin gel beads (gel core) dispersed in the liquid paraffin.
  • the obtained gelatin gel beads were washed several times with phosphate buffered saline to remove liquid paraffin.
  • HA solution containing 0.8% (w / v) of the hyaluronic acid derivative produced as described above, and 1100 U / mL of horseradish peroxidase (horse radish peroxidase: HRP, manufactured by Wako Pure Chemical Industries, Ltd.) HA / HRP solution was prepared by mixing the HRP solution to be mixed at a volume ratio of 10: 1 (hyaluronic acid solution: HRP solution).
  • the gelatin gel beads prepared as described above were suspended in the HA / HRP solution to prepare an HA / HRP solution containing the gelatin gel beads at 0.1 mL / mL. Further, 5 mL of hydrogen peroxide (35% (w / v), manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1 L of liquid paraffin, and the mixture was stirred for 30 minutes using a homogenizer. Then, liquid paraffin containing hydrogen peroxide was prepared by removing undissolved hydrogen peroxide solution by centrifugation.
  • hydrogen peroxide 35% (w / v)
  • a double tube structure was formed by placing a 26G syringe in a 21G syringe. Then, the gelatin gel bead-containing HA / HRP solution is flowed into the 26G syringe at a flow rate of 4.5 mL / h, and hydrogen peroxide-containing liquid paraffin is flowed between the 26G syringe and the 21G syringe at a flow rate of 4.2 mL / min. The gelatin gel beads-containing HA / HRP solution was allowed to flow out into the hydrogen peroxide-containing liquid paraffin from the tip of the 26G syringe. In this way, spherical composite gel beads composed of gelatin gel beads (gel core) and hyaluronic acid gel film (gel shell) covering the gelatin gel beads dispersed in hydrogen peroxide-containing liquid paraffin were formed.
  • the HA / HRP solution layer covering the gelatin gel beads is brought into contact with hydrogen peroxide-containing liquid paraffin so that the HA / HRP solution layer is cross-linked and gelled, and the hyaluronic acid gel film covering the gelatin gel beads is formed. Formed. The resulting composite gel beads were collected by centrifugation.
  • a solution containing the recovered composite gel beads was added to a culture solution (GM501, GYNEMED), and the composite gel beads were selected. That is, of the recovered composite gel beads, only the composite gel beads capable of visually recognizing the gel core of the gelatin gel beads were selectively collected using a thin glass pipette having an outer diameter of about 200 ⁇ m to 300 ⁇ m.
  • a hyaluronic acid capsule composed of a liquid core formed by dissolving gelatin gel beads and a hyaluronic acid gel film covering the liquid core was obtained as a sperm cryopreservation container.
  • the diameter of the HA capsule produced as described above was 219 ⁇ 18 ⁇ m (average diameter ⁇ standard deviation), and the thickness of the hyaluronic acid gel film was 30 ⁇ 7 ⁇ m (average thickness ⁇ standard deviation). .
  • the diameter of the HA capsule and the thickness of the hyaluronic acid gel film were measured by analyzing 100 HA capsule photographs taken with an optical microscope using image analysis software (ImageJ, manufactured by the National Institutes of Health, USA). did.
  • sperm cryopreservation As sperm to be cryopreserved in HA capsules, sperm collected from a human male patient but not used in human in vitro fertilization was used. That is, spermatozoa collected for in vitro fertilization but not used at Medical Corporation Sankeikai IVF Namba Clinic (Osaka, Japan) were used after informed consent was obtained and consent was obtained.
  • NARISIGE micromanipulation system equipped with a holding pipette (KITAZATO), an injection pipette (Sunlight medical), and a phase-contrast microscope used for microinsemination
  • the holding pipette was a glass pipette having an inner diameter of about 15 ⁇ m.
  • the injection pipette was a glass pipette having an inner diameter of about 4 ⁇ m.
  • sperm was injected into the liquid core of the HA capsule. That is, a holding pipette is operated under a phase-contrast microscope, and the HA capsule obtained as described above is placed in the tip of the holding pipette in a culture solution (Irvine) in a petri dish placed on a microscope stage. Retained.
  • a culture solution Irvine
  • the injection pipette was operated under a phase contrast microscope, and three spermatozoa were held inside the injection pipette in the culture solution (Irvine) in the petri dish placed on the microscope stage.
  • the injection pipette was operated under a phase contrast microscope, and three sperm were injected from the injection pipette into the liquid core in the HA capsule held by the holding pipette.
  • the tip of the injection pipette holding the sperm inside is inserted into the hyaluronic acid gel film of the HA capsule and penetrated, and the liquid from the tip of the injection pipette disposed in the liquid core of the HA capsule Sperm was released into the core.
  • an HA capsule holding three sperm was obtained.
  • a hollow capsule composed of a zona pellucida prepared from egg cells that were not fertilized after microinsemination was prepared.
  • egg cells collected from humans but not fertilized in microinsemination are treated with 17 ⁇ g / mL of cytochalasin B (Sigma Aldrich), and then the cytoplasm is removed by aspiration with an injection pipette. It was prepared by.
  • This egg cell was used after obtaining informed consent and consent from an egg cell collected for in vitro fertilization but not used at IVF Namba Clinic (Osaka Prefecture, Japan). did.
  • sperm was injected into the liquid core of the ZP capsule by the same operation as that of the above-mentioned HA capsule, thereby obtaining a total of 11 ZP capsules each holding three sperm.
  • HA capsules and ZP capsules holding sperm were stored frozen. That is, a plurality of droplets of 10 ⁇ L of a frozen solution containing 0.1 M sucrose-20% alternative serum (serum supplement) were formed on a 6 cm diameter cell culture dish. Then, each sperm-containing HA capsule or sperm-containing ZP capsule obtained as described above was suspended in one droplet one by one and incubated at room temperature for 5 minutes.
  • cryopreservation device (Cryotop (registered trademark), manufactured by KITAZATO CORPORATION)
  • a droplet of about 1.0 ⁇ L of the capsule-containing frozen solution after incubation is formed, and the droplet is placed on the liquid nitrogen surface.
  • Incubation was performed for 2 minutes while being exposed to liquid nitrogen vapor at a position 4.5 cm away from the substrate.
  • the cryopreservation instrument after incubation on liquid nitrogen vapor was cryopreserved for 24 hours or more in liquid nitrogen.
  • the frozen HA capsules and ZP capsules were thawed. That is, the cryopreservation instrument taken out from liquid nitrogen was incubated in a culture solution at 37 ° C. for 1 minute to thaw the HA capsule and ZP capsule in the cryopreservation instrument.
  • the capsule recovery rate, sperm recovery rate, and sperm motility rate were evaluated for each of the thawed HA capsules and ZP capsules. That is, for the HA capsule, first, a 10 ⁇ L droplet containing 40 IU / mL hyaluronidase was formed on a 6 cm diameter cell culture dish. Then, the thawed HA capsule was suspended in the droplet, and the droplet was coated with 5 mL of culture mineral oil (KITAZATO). Further, the HA capsule-containing droplets were incubated at 37 ° C. for 10 minutes to decompose the hyaluronic acid gel film (gel shell) of the HA capsules. Thereafter, the sperm retained in the HA capsule was collected using an injection pipette. Then, under the microscope, the number of sperm collected from the HA capsule and the number of sperm moving out of the collected sperm were counted.
  • KITAZATO culture mineral oil
  • the ZP capsule first, a 10 ⁇ L droplet of a culture solution (Irvine) was formed on a 6 cm diameter cell culture dish. Subsequently, the thawed ZP capsule was suspended in the droplet. Thereafter, sperm was sucked and collected from the ZP capsule using an injection pipette. Then, the number of sperm collected from the ZP capsule and the number of sperm moving out of the collected sperm were counted.
  • a culture solution Irvine
  • FIG. 3 shows the results of evaluating the capsule recovery rate, sperm recovery rate, and sperm motility rate for each of the HA capsules and ZP capsules after thawing. As shown in FIG. 3, all 11 capsules were recovered for each of the HA capsule and the ZP capsule.
  • sperm recovery rate was 90.9 ⁇ 15.5%.
  • ZP capsule 32 spermatozoa could be recovered, so that the sperm recovery rate was 97.0 ⁇ 10.0%. There was no statistically significant difference in sperm recovery between HA capsules and ZP capsules (t-test).
  • the sperm motility rate was 15.1 ⁇ 17.4%.
  • the ZP capsule out of the 32 sperm collected, movement of 7 sperm was confirmed, so the sperm motility rate was 21.2 ⁇ 16.8%. There was no statistically significant difference in sperm motility between HA capsules and ZP capsules (t-test).
  • a method for recovering sperm from the HA capsule As a method for recovering sperm from the HA capsule, a method of recovering sperm by decomposing the hyaluronic acid gel membrane of the HA capsule with hyaluronidase, and sperm by suction with an injection pipette without decomposing the hyaluronic acid gel membrane of the HA capsule was compared with the method of recovering. In all cases, the operation of the injection pipette was performed by an embryo culturer who was used to microinsemination.
  • HA capsules were produced in the same manner as in Example 2 described above. As sperm retained in the HA capsule, sperm collected from a human male patient but not used in human in vitro fertilization was used. That is, spermatozoa collected for in vitro fertilization but not used at the medical corporation Sankeikai IVF Namba Clinic (Osaka Prefecture, Japan) were used after obtaining informed consent and obtaining consent.
  • Example 2 3 sperm were injected into each of the 6 HA capsules to obtain 6 HA capsules each holding 3 sperm.
  • the resulting sperm-containing HA capsule was incubated at 37 ° C. for 15 minutes.
  • sperm was collected by enzymatic degradation of the hyaluronic acid gel membrane. That is, 3 ⁇ L of a suspension containing 3 sperm-containing HA capsules and 40 IU / mL hyaluronidase was prepared, and the resulting suspension was incubated at 37 ° C. for 10 minutes, whereby hyaluronic acid of the HA capsule was The gel film was broken down.
  • the ratio (%) of the number of sperm collected in the PVP to the number of sperm retained in the HA capsule was evaluated as a sperm collection rate.
  • the ratio (%) of the number of sperm in which motility was confirmed with respect to the number of sperm collected in PVP was evaluated as the sperm motility rate.
  • sperm was collected by an injection pipette without decomposing the hyaluronic acid gel membrane.
  • an injection pipette (a glass pipette having an inner diameter of about 4 ⁇ m) is inserted into a liquid core through a hyaluronic acid gel film, and the sperm in the liquid core is injected into the liquid core. Aspirated into pipette.
  • the sperm in the injection pipette was released into 10 ⁇ L of polyvinylpyrrolidone (PVP, molecular weight 360,000). And the ratio (%) of the number of the sperm collect
  • PVP polyvinylpyrrolidone
  • FIG. 4 shows the sperm recovery rate and sperm motility rate when sperm was recovered by enzymatic degradation of hyaluronic acid gel membrane and when sperm was recovered by injection pipette without decomposing the hyaluronic acid gel membrane. The evaluation results are shown.
  • motility was confirmed for two sperm out of the collected five sperm, but motility was not confirmed for three sperm, and the sperm motility rate was 40 0.0%.
  • the sperm recovery rate was 88.9% and the sperm motility rate was 100%.
  • the method for recovering sperm retained in the HA capsule is simpler, faster and more reliable than the recovery method using the injection pipette by the enzymatic decomposition of the hyaluronic acid gel membrane of the HA capsule. It was confirmed that it was remarkably superior.
  • a sperm storage container having a gel shell containing cellulose chains was produced. That is, first, similarly to the production of the hyaluronic acid derivative in Example 2 described above, a carboxymethyl cellulose (CMC) (400-800 cps-2%, aqueous solution at 25 ° C., manufactured by Sigma) has a phenol group as a crosslinkable functional group. CMC derivatives (crosslinkable CMC) were produced by chemical introduction. Specifically, CMC and tyramine hydrochloride were dissolved in 50 mM MES buffer (pH 6) to 1% (w / v) and 46 mM, respectively.
  • CMC carboxymethyl cellulose
  • tyramine hydrochloride were dissolved in 50 mM MES buffer (pH 6) to 1% (w / v) and 46 mM, respectively.
  • a CMC solution containing 1.0% (w / v) of CMC The sperm cryopreservation container is composed of a liquid core formed by dissolving gelatin gel beads (gel core) and a CMC gel film (gel shell) covering the liquid core. CMC capsules were obtained. The average diameter of the CMC capsules thus produced was 156 ⁇ m, and the average thickness of the CMC gel film was 25 ⁇ m.
  • a sperm cryopreservation container composed of a liquid core and an alginate gel film (gel shell) covering the liquid core was manufactured. That is, gelatin gel beads were dispersed in a 0.6% (w / v) sodium alginate solution or a 1.0% (w / v) sodium alginate solution. Next, the gel solution is coated with gelatin gel beads (gel core) and the alginate gel by dropping the resulting solution from a syringe with a 26G syringe needle into a 100 mM calcium chloride aqueous solution using electrostatic force. Spherical composite gel beads composed of a membrane (gel shell) were formed.
  • an injection pipette was passed through the gel shell. It was examined whether it could be inserted into the liquid core. In all cases, the operation of the injection pipette was performed by an embryo culturer who was used to microinsemination.
  • the injection pipette could not penetrate through the gel shell (alginate gel film). That is, the injection pipette pierced in the gel shell of the AG capsule is bent before the tip portion penetrates the gel shell and cannot be used.
  • the injection pipette could be penetrated through the gel shell (CMC gel film) of three CMC capsules out of the five CMC capsules.
  • CMC capsules can also inject sperm into the liquid core using an injection pipette.
  • the HA capsule was able to inject sperm more efficiently and reliably than the CMC capsule.
  • a sperm storage container composed of a liquid core and an agarose gel membrane (gel shell) covering the liquid core was manufactured. That is, first, in the same manner as in the production of the hyaluronic acid derivative in Example 2 above, alginic acid (sodium alginate, I-1G, manufactured by Kimica) was chemically introduced with a phenol group as a crosslinkable functional group to thereby obtain alginic acid. A derivative (crosslinkable alginic acid) was prepared. Specifically, alginic acid and tyramine hydrochloride were dissolved in 50 mM MES buffer (pH 6) so as to be 0.5% (w / v) and 4.5% (w / v), respectively.
  • Example 2 similarly to Example 2 described above, a double tube structure was formed by placing a 26G syringe in a 21G syringe. Then, similarly to Example 2 described above, CF-KRH is allowed to flow through the 26G syringe at a flow rate of 4.5 mL / h, and is prepared in the same manner as Example 2 described above between the 26G syringe and 21G syringe.
  • the hydrogen peroxide and lecithin-containing liquid paraffin obtained by adding 3% (w / w) of lecithin to the hydrogen peroxide-containing liquid paraffin were flowed at a flow rate of 4.2 mL / min, and the CF-KRH was The liquid paraffin was allowed to flow out from the tip of the 26G syringe.
  • spherical alginate gel beads gel core
  • the resulting alginate gel beads were then collected by mixing with CF-KRH and then centrifuging.
  • agarose solution of 40 ° C. was prepared by dispersing agarose (manufactured by Sigma, Low gelling temperature) at 4.0% (w / v) in CF-KRH and dissolving it by heating in a microwave oven. .
  • an agarose solution containing the alginate gel beads was prepared by mixing the alginate gel beads produced as described above and the 40 ° C. agarose solution at a volume ratio of 1:20 (alginate gel beads: agarose solution). .
  • a flow rate of agarose solution containing alginate gel beads in the 26G syringe is 4.5 mL / h.
  • a liquid paraffin containing 3% (w / w) lecithin was allowed to flow between the 26G syringe and the 21G syringe at a flow rate of 4.2 mL / min, and the agarose solution containing the alginate gel beads was added to the 26G syringe. It was made to flow out into the lecithin containing liquid paraffin from the tip of the syringe.
  • the recovered composite gel beads are held in CF-KRH containing 0.2 mg / mL alginate lyase for 1 hour, and the alginate gel beads inside the composite gel beads are decomposed to obtain the sperm cryopreservation container.
  • the agarose capsule comprised from the liquid core produced
  • the diameter of the agarose capsule thus produced was about 150 ⁇ m, and the thickness of the agarose gel membrane was 10 ⁇ m to 30 ⁇ m.
  • agarose capsule-containing CF-KRH was prepared by dispersing about 200 agarose capsules produced as described above in 0.2 mL of CF-KRH.
  • 30 U / mL of a commercially available agarase (Thermostable ⁇ -Agarase, manufactured by Nippon Gene Co., Ltd.) was added to 0.2 mL of this agarose capsule-containing CF-KRH.
  • CF-KRH containing an agarose capsule and agarase was incubated at 37 ° C. for 5 hours.
  • the agarase used exhibited maximum activity at 50 ° C to 60 ° C, but it was obtained from the manual (http://www.nippongene.com/pdf/manual/man_Thermostable_B_Agarase_ver4.pdf). According to (possible), the activity was about half of the maximum activity even at 37 ° C. Further, the manual described that 6 ⁇ U of agarase is used for 200 mg of agarose gel block, and that the agarose gel block is degraded in 10 minutes at 50 ° C. to 60 ° C.

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Abstract

L'invention concerne : un récipient destiné à être utilisé pour la cryoconservation de sperme, qui permet d'effectuer le recueil et la cryoconservation de sperme de manière efficace même lorsque le nombre des spermatozoïdes est faible ; un procédé de production du récipient ; un procédé de cryoconservation de sperme ; et un procédé de fécondation in vitro. Le procédé de cryoconservation de sperme selon un mode de réalisation de la présente invention comprend les étapes (a) à (f) suivantes : (a) l'utilisation d'un récipient (10) qui est pourvu d'une coque en gel (11) contenant une chaîne de glycosaminoglycane et/ou une chaîne de cellulose et d'un noyau liquide (12) appliqué sur la coque en gel (11) ; (b) l'injection des spermatozoïdes (S) dans le noyau liquide dans le récipient afin d'obtenir le récipient contenant les spermatozoïdes à l'intérieur ; (c) la réalisation de la cryoconservation du récipient contenant les spermatozoïdes à l'intérieur ; (d) la décongélation du récipient cryoconservé ; (e) le traitement de la coque en gel dans le récipient décongelé par une enzyme apte à décomposer la chaîne de glycosaminoglycane et/ou une enzyme apte à décomposer la chaîne de cellulose pour décomposer au moins une partie de la coque en gel ; et (f) le recueil des spermatozoïdes à partir du récipient après la décomposition de la coque en gel.
PCT/JP2016/070194 2015-07-07 2016-07-07 Récipient destiné à être utilisé pour la cryoconservation de sperme et procédé de production correspondant, procédé de cryoconservation de sperme, et procédé de fécondation in vitro WO2017007006A1 (fr)

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